Modification of pertussis toxin

ABSTRACT

The three-dimensional structure of crystalline pertussis holotoxin (PT) has been determined by X-ray crystallography. Crystal structures have also been determined for complexes of pertussis toxin with molecules relevant to the biological activity of PT. These three-dimensional structures were analyzed to identify functional amino acids appropriate for modification to alter the biological properties of PT. Similar procedures may be used to predict amino acids which contribute to the toxicity of the holotoxin, to produce immunoprotective, genetically-detoxified analogs of pertussis toxin.

REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/292,968 filed Aug. 22,1994, now abandoned which is a continuation-in-part of U.S. patentapplication Ser. No. 08/251,121 filed May 31, 1994, now abandoned whichitself is a continuation-in-part of U.S. patent application Ser. No.110,947 filed Aug. 24, 1993, now abandoned.

FIELD OF INVENTION

The present invention relates to a method for the prediction offunctional amino acid residues in pertussis toxin, in order tomanipulate the biological properties of the toxin, by determination ofand examination of the crystal structures of the toxin alone and ofcomplexes of the toxin with molecules relevant to its biologicalactivity, including carbohydrate ligands, nucleotide effectors andsubstrates.

BACKGROUND OF THE INVENTION

Whooping cough, or pertussis, is a severe, highly contagious respiratorydisease of infants and young children caused by infection withBordetella pertussis. Owing to the many virulence factors associatedwith this organism, the pathogenesis of the disease is still not fullyunderstood; however, it is generally recognized that major systemiceffects are caused by pertussis toxin (PT). This material exhibits awide range of biological activities, as illustrated by such alternativenames as lymphocytosis-promoting factor, histamine-sensitizing factorand islet-activating protein (ref. 1--a list of the references appearsat the end of the disclosure, each of which reference is incorporatedherein by reference thereto).

PT is a 105-kDa exotoxin encoded by the tox operon and consists of fivepolypeptide subunits (S1 to S5) arranged in an A-B structure typical ofsome bacterial toxins. The S2, S3, S4, (two copies) and S5 subunits forma pentamer (the B oligomer) which when combined with the S1 subunitforms the holotoxin. S1 is an enzyme with ADP-ribosyltransferase andNAD-glycohydrolase activities. Its natural function is to catalyse thetransfer of the ADP-ribose portion of nicotinamide adenine dinucleotide(NAD) to the membrane-bound guanine nucleotide-binding negativeregulatory G-protein of adenylate cyclase (G_(i)), resulting in anincrease in cyclic-AMP synthesis. This activity, which is the primarycause of PT toxicity, can be conveniently examined in vitro using assubstrate the retinal G-protein transducin, which is a close analogue ofG_(i) (ref. 2). Such studies have demonstrated that PT is activated byadenine nucleotides, in particular by adenosine triphosphate (ATP)(refs. 3,4), while S1 itself is active only in the presence of thiols,such as dithiothreitol, that are required to reduce the single disulfidebond of S1 (ref. 5).

The B oligomer mediates the binding of the holotoxin to target cells andfacilitates entry of the A protomer. PT has lectin-like properties,binding to glycoconjugates on many cell surfaces and to theoligosaccharide moieties of many serum glycoproteins (refs. 6,7). It hasbeen reported that the toxin preferentially recognizes asparagine-linkedoligosaccharide chains containing (2α-6)-linked sialic acid residues(ref. 6). However, a number of complex carbohydrate sequences are bound,and there is evidence that PT contains at least two binding domains withdifferent specificities on each of the subunits S2 and S3 (refs. 7,8).

Several studies have indicated that PT is a major protective antigenagainst pertussis. Thus, purified, toxoided PT protects mice againstboth intracerebral and respiratory challenges with B. pertussis (refs.9,10). Polyclonal anti-PT antisera and some anti-PT monoclonalantibodies also protect against challenge (refs. 9,10,11). Furthermore,a mono-component pertussis vaccine containing chemically toxoided PTshowed efficacy in a human clinical trial (ref. 12).

Defined whooping cough vaccines have been produced by the isolation ofantigens from cultures of B. pertussis. Of the antigens present inacellular vaccines, only PT is toxic. Detoxification of PT has beenperformed by non-specific chemical modification with formaldehyde,glutaraldehyde, hydrogen peroxide, tetranitromethane and ethyleneimine.Treatment of PT with formaldehyde results in a reduction inimmunogenicity and the loss of important protective epitopes, andhydrogen peroxide and tetranitromethane detoxification processes havebeen shown to significantly reduce the immunogenicity of the molecule.Furthermore, prolonged treatment with glutaraldehyde results inwhole-cell pertussis vaccines with low potency. Of further concern isthe reversion of formalin-inactivated PT toxoids to toxicity. However,PT can be irreversibly detoxified with appropriate concentrations ofglutaraldehyde (ref. 13). Such problems of reduced immunogenicity andresidual toxicity have been addressed by genetically manipulating thetox operon to produce inactivated PT analogs (refs. 14,15).

The tox operon has been cloned and sequenced from several strains of B.pertussis, and consists of a single promoter and a polycistronicarrangement of the subunit genes in the order S1, S2, S4, S5 and S3. Toremove the enzymatic activity of S1, functional amino acids within thesubunit were proposed on the basis of biochemical studies or sequencecomparisons with other bacterial toxins and subjected to in vitromutagenesis. Truncated S1 proteins were produced in Escherichia coli andused to demonstrate that the amino terminus of S1 is required forenzymatic activity. An important region was located between Tyr-8 andPro-14 with an amino acid sequence similar to sequences in cholera toxin(CT) and E. coli heat-labile toxin (LT) (refs. 16,17). Amino acids inthis region that contribute to the ADP-ribosyltransferase activity of PTwere identified by substitution mutagenesis. In particular, the Arg-9 toLys-9 replacement was found to greatly reduce enzymatic activity (ref.18). A second region of S1, located between Val-51 and Tyr-59, is alsoconserved in CT and LT. This region was also mutated and some of theresidues were shown to be involved in the toxicity of PT, includingSer-52 and Arg-58 (refs. 15,19). The glutamic acid residue at position129 in the S1 subunit was identified as a residue involved in catalysisor NAD binding (ref. 20), and substitution at this site resulted in asubstantial reduction in enzymatic and toxic activities. PT has alsobeen detoxified by mutating Trp-26, His-35 and Cys-41 (ref. 15).

Pertussis toxin has also been detoxified by modification of its cellbinding properties, for example by deletion of Asn-105 in the S2 subunitand Lys-105 in the S3 subunit, and by substitution of the Tyr-82 residuein S3 (refs. 21,22). However, the characteristics of the carbohydratebinding sites are imperfectly understood, and a definitive applicationof this approach has not yet been achieved. Since the molecularmechanism by which PT exerts its various biological activities are stillnot completely understood, other methods for identifying functionalamino acid residues are also useful.

One such method of the present invention is based upon examination ofthe three-dimensional (3D) structure of PT. A useful embodiment of thisapproach is to relate previously determined features of the functionalsites of PT to the observed structural geometry in order to providegreater insight into the underlying molecular mechanisms. This permitsthe rational mutation of PT at preselected sites to maximize (forexample) detoxification but retain immunogenicity. In particular, itallows for modifying PT at sites differently involved in the biologicalactivity of PT. Another embodiment of this approach is to compare the 3Dstructure of PT with those of other bacterial toxins with somefunctional and/or structural resemblance to PT. These include diphtheriatoxin (DT) (ref. 23), Pseudomonas exotoxin A (ETA) (refs. 24,25), theheat-labile toxin of E. coli (LT) (refs. 27,28) and verotoxin-1 (VT)(ref. 29).

A particularly useful application of the crystallographic method is toexamine the 3D structure of crystalline complexes of PT with moleculesrelevant to its biological activity. In this way, the amino acidresidues of PT responsible for interaction with such ligands can bedetermined by direct inspection, allowing rational strategies to bedeveloped for their replacement or modification in order to alter thebiological activities of PT.

Suitable examples of such molecules are carbohydrates representing thenatural ligands for PT found as components of cell-surfaceglycoconjugates. Some of the characteristics of PT-binding glycosylchains have been determined by direct binding studies of PT or PTsubunits to glycoproteins, glycolipids and cell surfaces (refs. 6,7,8),or by competitive inhibition of the binding or biological activity of PTby small oligosaccharides (refs. 6,7,30). Examples of oligosaccharidesthat might be expected as a result of such work to form definedcomplexes with PT are shown in Table 1 below (The Tables appear at theend of the disclosure). Once the amino acids responsible for interactionwith these ligands have been identified, they may be modified (forexample, by mutagenesis) to enhance or diminish this interaction andthereby alter the biological activities of PT.

Other examples of functionally relevant PT binding molecules areeffectors, such as ATP, and substrates, such as NAD, transducin or otherG-protein. Since synthetic peptides representing the C-terminal 20 aminoacids of the α-subunits of transducin and other G-proteins have beenshown to be substrates for the ADP-ribosyltransferase activity of PT,these molecules are also candidates for the generation of crystallinecomplexes with PT. In so far as NAD itself can be hydrolysed by PT, itmay be preferable to seek a non-hydrolysable or poorly hydrolysed analogof NAD for the purpose of forming a stable complex amenable to X-raycrystallography. Alternatively, the study can be performed using a PTanalog with inherently low catalytic activity, such as that in which theGlu-129 residue of S1 has been replaced by Gly (ref. 15). Moreover,since ligands are expected to bind to defined regions of the proteinsurface, it may not be necessary to employ the holotoxin in every case.For example, information on the binding sites of NAD or transducin maybe obtained by examining the crystal structure of complexes with theisolated S1 subunit.

SUMMARY AND GENERAL DESCRIPTION OF INVENTION

In accordance with one aspect of the present invention, there isprovided a method of predicting at least one site contributing to thebiological activity of pertussis holotoxin, which comprises analyzing athree-dimensional structure of crystalline pertussis holotoxindetermined by X-ray crystallography in relation to known informationconcerning protein structure and function to identify the at least onesite.

Such a biological activity of pertussis holotoxin may include toxicity,cell-binding, mitogenicity, enzymatic activity and adjuvanticity of thepertussis holotoxin. The at least one site which is predicted by themethod provided herein may comprise a single amino acid or a sequence ofamino acids.

Such analyzing step may comprise comparing the three-dimensionalstructure of pertussis holotoxin with known three-dimensional structuresof enzymes with substantial functional resemblance to pertussisholotoxin (including bacterial toxins having ADP-ribosyl transferaseactivity), and identifying structurally conserved regions between thepertussis holotoxin and the enzymes.

Such analyzing step also may comprise comparing the three-dimensionalstructure of pertussis holotoxin with known three-dimensional structuresof other proteins with carbohydrate binding properties, and identifyingregions of structural resemblance of the pertussis holotoxin to theproteins.

In these procedures, analysis may also be effected by aligning aminoacid sequences of pertussis holotoxin with those of the enzymes orproteins with carbohydrate binding properties, as the case may be,according to structural equivalence (i.e., the strucurally conservedregions or regions of structural resemblance, respectively) determinedby the identification step.

The analyzing step further may comprise locating, within thethree-dimensional structure of pertussis holotoxin, amino acid residuesknown to contribute to the biological activity of the holotoxin, andidentifying spatially-proximate amino acid residues interacting withsaid known amino acid residues within said three-dimensional structure.

Following identification of the at least one site by the procedureprovided herein, the identified at least one site may be modified toalter the biological activity of the pertussis holotoxin, whichmodification may be effected by genetic, chemical or biochemical means.

Accordingly, the present invention includes the use of thethree-dimensional structure of crystalline pertussis holotoxindetermined by X-ray crystallography for identifying at least one site inthe pertussis holotoxin molecule contributing to the biologicalactivity, including any of the activities noted above.

In accordance with a further aspect of the present invention, there isprovided a method of identifying at least one site in pertussisholotoxin that interacts with a molecule that is capable of forming acomplex with the holotoxin, the method comprising:

(a) providing a crystalline complex between at least a portion ofpertussis holotoxin and the molecule;

(b) determining the three-dimensional structure of the complex by X-raycrystallography; and

(c) analysing the structure to identify the at least one interactingsite.

The at least one identified site may contribute to toxicity, cellbinding, mitogenicity, enzymatic activity or adjuvanticity of thepertussis holotoxin.

The at least a portion of the holotoxin with which the complex is formedmay be the entire pertussis holotoxin, an analog thereof, a subunit ofthe holotoxin, a portion of a subunit, or a combination of subunits.

The step of forming the complex between the molecule capable of forminga complex with the holotoxin and the at least a portion of the holotoxinmay comprise exposing crystals of the at least a portion of theholotoxin under conditions to effect formation of the crystallinecomplex without substantial disruption of the crystals.

The molecule capable of forming a complex with the holotoxin may be aligand, such as a cell-surface ligand, including carbohydrates, such asa glycolipid or a glycoprotein, an effector molecule, such as an adeninenucleotide, including ATP, or a substrate for the enzymatic activity ofPT. Such substrates include NAD and analogs of NAD that are notsubstantially hydrolysable by pertussis toxin. The substrate may also bea GTP-binding protein (a G-protein), an α-subinit of a GTP-bindingprotein or a C-terminal fragment of an α-subunit of a GTP-bindingprotein. Convenient GTP-binding proteins include G_(i), G_(o) andtransducin.

Following identification of the at least one site by the procedureprovided herein, the identified at least one site may be modified, forexample, by genetic, biochemical, or chemical means, to alter abiological activity, such as toxicity, enzymatic activity, mitogenicitycell-binding and adjuvanticity, of the pertussis holotoxin.

The at least one identified site may be at least one amino acid and maybe modified by effecting mutagenesis of a tox operon encoding theholotoxin to remove or replace a nucleotide sequence coding for said atleast one amino acid residue and to produce a mutant tox operon, andexpressing the mutant tox operon in a Bordetella organism to produce themodified holotoxin.

The present invention further comprises a crystalline form of isolatedpertussis holotoxin in which the molecules of pertussis toxin have thethree-dimensional structure represented by FIGS. 1 and 2 describedbelow. The crystalline form of pertussis holotoxin may be in dimericform, as shown in FIG. 8, described below. The crystalline form ofpertussis holotoxin may have a space group P2₁ 2₁ 2₁ with celldimensions a=163.8 Å, b=98.2 Å and c=194.5 Å. The crystalline form ofpertussis holotoxin may be in the form of a complex with a moleculecapable of forming a complex with the holotoxin, as described in detailabove. Specific examples of such complexes may have thethree-dimensional structure represented by FIGS. 10 and 11, describedbelow, or by FIGS. 13 and 14, described below.

In addition, the present invention includes a crystalline form ofisolated pertussis holotoxin characterized by atomic co-ordinatesspecified in accession no. 1 PRT of the Brookhaven Protein Data Bank,Brookhaven, N.Y. USA.

The provision of a crystalline form of pertussis holotoxin allows acomparison with other proteins having functional resemblance topertussis holotixin (for example bacterial toxins from Campylobacterjejuni and Clostridium botulinum) with an aim to beneficially modifyingsuch other proteins. For example, bacterial toxins are frequentlyprotective immunogens but require detoxification before they can be usedas immunogenic compositions. The ability to identify currently unknownsites that contribute to toxicity of such toxins by a comparison withthe three dimensional structure of pertussis holotoxin provides atechnique for detoxification of such toxins to provide usefulimmunogenic but non-toxic analogues.

The crystalline form of pertussis holotoxin as provided herein is of aparticularly high purity and is useful as a primary standard formeasuring the quantity, purity or efficacy of less pure compositionscontaining PT.

The present invention further includes a method for the production of amodified pertussis holotoxin, which comprises (a) identifying at leastone amino acid residue of the holotoxin for modification by utilizingthe prediction procedure provided herein; (b) effecting mutagenesis of atox operon encoding the holotoxin to remove or replace a nucleotidesequence coding for the at least one amino acid residue and to produce amutant tox operon; and (c) expressing the mutant tox operon in aBordetella organism to produce the modified holotoxin.

In an additional aspect of the invention, there is provided a method forproducing a modified form of at least a portion of pertussis holotoxin,comprising (a) forming a crystalline complex between at least a portionof pertussis holotoxin and a molecule capable of complexing with theholotoxin; (b) determining a three-dimensional structure of the complex;(c) analysing the structure to identify at least one amino acid residueof the at least a portion of pertussis holotoxin interacting with themolecule; (d) effecting mutagenesis of a nucleotide sequence encodingthe at least a portion of the pertussis holotoxin to remove or replace acodon for the at least one amino acid and/or to insert at least onecodon adjacent the codon for the at least one amino acid to produce amutant nucleotide sequence; and (e) expressing the mutant nucleotidesequence to produce the modified form of at least a portion of pertussisholotoxin. The at least a portion of pertussis holotoxin may bepertussis holotoxin or an analog thereof and the step of expressing themutant nucleotide sequence is effected in a Bordetella organism.Alternatively, the at least a portion of pertussis holotoxin may be asubunit of the holotoxin, a portion of such a subunit or a combinationof subunits. The at least one additional codon may be inserted adjacentthe 3'-codon of the nucleotide sequence encoding the S1 subunit, inparticular a codon coding for a negatively-charged amino acid, forexample, Asp or Glu.

The identified amino acid residue in these procedures for producingmodified products may comprise at least one amino acid residue whichcontributes to a biological activity, which may comprise toxicity,cell-binding, mitogenicity, enzymatic activity and adjuvanticity.

The at least one amino acid residue which is mutated may comprise one ofthose residues as specified in Table 3 below and the substitution madealso may be as set forth in Table 3. The at least one amino acid residuealso may be located from residue 184 to 203 or from residue 211 to 220of subunit S1 of the holotoxin.

The invention additionally includes a mutant pertussis holotoxin whereinat least one amino acid residue in the S1, S2, S3, S4 or S5 subunits issubstituted by another amino acid residue or deleted, provided by theprocedure described herein. The at least one amino acid residue which ismodified in this aspect of the invention is listed in Table 3 below.Specific amino acid residue substitutions in the S1, S2, S3, S4 and S5subunits also are described in Table 3 below.

Particular embodiments of the invention include PT analogs havingmutations S2 Lys-83→Ala, S2 Arg-125→Ala and S3 Arg-125→Ala. Theactivities of such analogs are shown in Table 6 below.

The mutant pertussis holotoxin may comprise modification of at least oneamino acid residue located from 184 to 203 or located from 211 to 220 ofsubunit S1 of the holotoxin. The modification in residues 184 to 203 maybe effected to render the same more hydrophilic while the modificationin residues 211 to 220 may be effected to eliminate recognition byproteolytic enzymes.

The mutant pertussis holotoxin provided herein may further comprise atleast one additional amino acid provided at the C-terminal end of the S1subunit, particularly a negatively-charged amino acid, particularly Aspor Glu.

The present invention also extends to nucleic acid molecules encodingthe PT analogs as provided herein.

BRIEF DESCRIPTION OF DRAWINGS

The file of this patent application contains at least one drawingexecuted in color, namely FIGS. 1, 2, 6, 10, 11 and 13. Copies of thispatent with color drawing(s) will be provided by the Patent andTrademark Office upon request and payment of the necessary fee.

FIG. 1 shows a schematic representation of the B-oligomer of pertussistoxin viewed along the pseudo-5-fold axis from the side opposite to S1.Subunit S2 is shown in pale blue, S3 in dark blue, S4 in red, and S5 inyellow;

FIG. 2 shows a schematic representation of pertussis toxin viewedperpendicular to the 5-fold axis of the B-oligomer. Subunit S1 is shownin green, and the other subunits as in FIG. 1;

FIG. 3 shows a schematic representation of the active site of subunit S1of pertussis toxin with individual amino acid residues identifiedaccording to the standard one-letter coding system;

FIG. 4 shows a sequence alignment of structurally equivalent residues inthe active sites of four ADP-ribosylating toxins, namely PT, LT, ETA andDT (SEQ ID NOS: 1 to 20);

FIG. 5 shows a sequence alignment of the different PT B-subunits (S4, S5and the C-terminal parts of S2 and S3 (respectively SEQ ID NOS: 24, 25,22 and 23)) and the B-subunits of LT and VT (respectively SEQ ID NOS: 21and 26);

FIG. 6 shows a schematic representation of the N-terminal 93 residues ofS3 (left) and rat mannose binding protein (MBP) (right);

FIG. 7 shows a sequence alignment of 47 structurally equivalent residuesin MBP (SEQ ID NOS: 39 to 44) and the N-terminal domains of S2 and S3(respectively SEQ ID NOS: 27 to 32 and 33 to 38);

FIG. 8 shows a Cα tracing of the dimeric form of PT existing in thecrystal;

FIG. 9 is a stereo view showing the difference electron density for abiantennary undecasaccharide from human serum transferrin bound tosubunit S3 of PT;

FIG. 10 shows a schematic representation of the complex between PT and abiantennary undecasaccharide from human serum transferrin in the sameorientation as FIG. 1. Subunit S2 is shown in pale blue, S3 in darkblue, S4 in red, S5 in yellow and the carbohydrate in mauve;

FIG. 11 shows a schematic representation of the complex between PT and abiantennary undecasaccharide from human serum transferrin in the sameorientation as FIG. 2. Subunit S1 is shown in green, and the othersubunits and the carbohydrate as in FIG. 10;

FIG. 12 is a stereo view showing the binding to PT subunit S2 of theterminal NeuAc(2α,6)Gal moiety of a biantennary undecasaccharide fromhuman serum transferrin. This Figure depicts the sugar in relation tothe Cα trace of S2 and details of the binding site, with dashed linesindicating hydrogen bonds between the sugar (thick lines) and protein(thin lines);

FIG. 13 is a schematic representation of the complex between PT and ATP,demonstrating the electron density difference between the complex andthe holotoxin alone. This electron density difference, shown in lightblue, is largely attributed to the bound ATP molecule. Subunit S1 isshown in green and the B-oligomer in violet;

FIG. 14 shows a schematic representation of the complex between PT andATP, viewed approximately along the pseudo-5-fold axis. The helices andstrands common to the five B-subunits are depicted as ribbons and arrowsrespectively. The N-terminal domains of subunits S2 and S3 and the chainfragments connecting the secondary structural elements are depicted aslines. The C-terminus of subunit S1 (residues 226-235) and the ATPmolecule are located at the center of the figure and are drawn as asmooth coil and as a ball-and-stick model, respectively. All subunitsare labeled on their N-termini; and

FIG. 15 is a stereo diagram of the ATP binding site, showing the mostimportant residues that interact with the ATP molecule. The residuelabels comprise the residue number preceded by a character to indicatein which subunit the residue resides (A, B, C, D, E and F denote S1, S2,S3, S4a, S4b and S5, respectively);

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Example 1

This Example describes the crystallization of pertussis toxin (PT), datacollection and phase determination.

PT was purified from culture supernatants of B. pertussis strain 10536(ref. 8), and crystallized in space group P2₁ 2₁ 2₁ with cell dimensionsa=163.8 Å, b=98.2 Å, c=194.5 Å, by a modification of conditionsdescribed by Spangler et al. (ref. 32). PT crystals have also beenreported by Raghavan et al. (ref. 33). According to the modifiedconditions, stock solutions of PT in 0.1 M K-phosphate, pH 8.0, 50% v/vglycerol were stored at -20° C., and samples (1.2 to 2.0 mg/ml) weredialysed against buffer 1 (25 mM Na/K-phosphate, 0.25 M KCl, 0.02% NaN₃,pH 8.0). Seed crystals were grown by the hanging drop method,equilibrated against a precipitant of 0.3 to 0.5 M KCl in buffer 1.Macroseeds were transferred to protein drops equilibrated against 0.26to 0.35 M KCl in buffer 1 and typically grew to a size of 0.3 mm×0.2mm×0.15 mm. Diffraction data were collected at room temperature using asynchrotron radiation source (λ=1.04 Å) and image plate detector on theWeissenberg camera (ref. 34). The data were processed using WEIS (ref.35) and programs in the CCP4 package (obtained from DaresburyLaboratory, Daresbury, Warrington WA4 4AD, England). The x-raydiffraction data obtained is summarized in Table 2 below, and nowdescribed in detail.

"Native" data: Initially, the best data set (native 1) was collected to3.3 Å resolution from a crystal soaked in 20 mM trimethyl lead acetatefor 14 days, although these data do not differ significantly from truenative data. Later, a 2.9 Å data set (native 2) was collected thatmerged poorly with native 1 and the derivative data. The native 2 datawere collected from a crystal that had been soaked in 30 mM NAD for 3days, although difference Fourier analyses show no evidence of NADbinding.

Heavy atom derivatives: True native crystals were soaked respectively insaturated d-μ-iodobis-(ethylenediamine) diplatinum(II) nitrate (PIP) for7 days, saturated (NH₃)₂ Pt(NO₂)₂ for 7 days, 1 mM KIrCl₆ for 3 days, 1mM KAu(CN)₂ for 5 days, 1 mM OsCl₃ for 3 days.

Multiple isomorphous replacement (MIR) phase determination: Seven heavyatom sites were identified in the PIP derivative with SHELXS (ref. 36)using native 1 as native data. Parameters for these sites were refinedwith MLPHARE (ref. 37), and further sites in the PIP and otherderivatives were found by iterative use of difference Fourier analysesand heavy atom refinement. The overall figure of merit was 0.44 (25-3.5Å).

Density modification: The MIR phases were improved by solvent flatteningwith the DEMON density modification package (obtained from Dr. F. M. D.Vellieux, IBS/LCCP, 41 Ave. des Martyrs, 38027 Grenoble, Cedex 1,France), using a conservative solvent fraction of 50%. The resulting mapshowed a clear solvent boundary outlining two holotoxin molecules in theasymmetric unit. The relative orientation of the two molecules wasdetermined by a domain rotation function (ref. 38), using structurefactors calculated in space group P1 from spheres of densitycorresponding to each molecule. The translation needed to superimposerotated density from one molecule onto the density sphere for the secondmolecule was calculated with a phased translation function (ref. 38,39).An envelope around the two toxin molecules was calculated from a localcorrelation map (ref. 40), enclosing 44% of the asymmetric unit.Two-fold averaging and solvent flattening were carried out using theDEMON package, with gradual phase extension from 5.0 to 3.5 Å.

The atomic co-ordinates of the crystalline pertussis holotoxin asdetermined herein have been deposited as Accession Number 1 PRT of theBrookhaven Protein Data Bank, Brookhaven, N.Y., USA.

Example 2

This Example describes model building of the pertussis toxin molecule.

Interpretation of the electron density map of PT was facilitated by therecognition of similarity with known toxin structures, which helped todefine the secondary structure connections and chain direction in alarge part of the map. A molecular model of the A1-subunit of LT waspositioned in the density corresponding to S1 using a domain rotationfunction and phased translation function. In this way, the N-terminalthree-quarters of S1 was shown to resemble the A1-subunit of LT. Fiveseparate VT B-monomers were placed in the central part of the B-oligomerdensity using the program O (ref. 41). The central part of theB-oligomer was shown to share the fold common to VT and LT. Theperipheral domains of the B-oligomer (formed by the N-terminal portionsof S2 and S3) were built without reference to a known structure. Apartial model of one PT molecule was built using O, based on askeletonised map and guided by the positioned models. The structure ofthe second PT molecule of the asymmetric unit was generated using thenon-crystallographic symmetry operation. Subunits S2 and S3 were easilydistinguished by differences in side-chain electron density, especiallyat positions 26 (Lys/Gly), 50 (Arg/Pro), 65 (Gly/Gln), 143 (Trp/Arg) and148 (Arg/Ala).

The initial PT model, containing 85% of the residues with 5% fitted asalanines, was refined by rigid body refinement and energy minimizationusing X-PLOR (ref. 42). Phases from the refined model were combined withthe original MIR phases using SIGMAA (ref. 43). Errors in the initial PTmodel were clearly shown in the map calculated with the combined phases.Iterative cycles of model building, simulated annealing refinement withX-PLOR (including a non-crystallographic symmetry restraint), andcombination of model and MIR phases resulted in a model that was 98%complete. The 3.3 Å model was then refined against the 2.9 Å data set(native 2) using rigid body refinement and simulated annealing inX-PLOR. Further model building and refinement were carried out with thehigher resolution data.

This PT model contains 1868 amino acid residues (934 in each molecule,comprising 224/235 in S1, 196/199 in S2 and S3, 110/110 in both copiesof S4, 98/99 in S5) with no solvent molecules. The electron density iscompatible with the amino acid sequence from B. pertussis strain 10536(ref. 44). Missing residues are in regions of poor density at theN-termini of S1 (one residue), S2 (three residues), S3 (three residues)and S5 (one residue), and ten residues (211-220) near the C-terminus ofS1. Schematic representations of the B-oligomer and the holotoxin areshown in FIGS. 1 and 2.

The model includes restrained individual isotropic temperature factors.It has a crystallographic R-factor of 19.5% for all reflections between10.0 and 2.9 Å and tightly restrained geometry. The rms deviation fromideality is 0.014 Å for bond distances and 1.8° for bond angles. In theRamachandran plot of each molecule, 82% of the residues are in the mostfavoured regions and none are in disallowed regions (refs. 45). The rmsdifference in coordinates of all main chain atoms after superposition ofthe two molecules is 0.21 Å.

Example 3

This Example shows the analysis of the structure of pertussis toxin.

The overall structure of the PT molecule, which contains six separatepolypeptide chains, is shown schematically in FIGS. 1 and 2, whichemphasize the considerable topological complexity of PT. They depict theB-oligomer viewed along the 5-fold axis from the side opposite to S1 andthe holotoxin viewed perpendicular to the 5-fold axis of the B-oligomer.In these and other figures, α-helices are shown as spirals, β-strands asthick arrows and less-regular segments of the polypeptide chains as thinfilaments. Most details of the individual amino acid residues have beenomitted for simplicity.

The structure of PT displays some striking similarities with otherbacterial toxins, but also some significant differences which haveimportant implications for the functional activity of PT. FIG. 1 showsthe planar triangular arrangement of the B-oligomer. The polypeptidechain folds of S4, S5 and the C-terminal domains of S2 and S3 areremarkably similar to each other and to the corresponding subunits of VTand LT. The presence of a central pore with approximate 5-fold symmetryis also characteristic of VT and LT. The N-terminal domains of subunitsS2 and S3, which constitute the acute apices of the triangulararrangement, are unique to PT and therefore of particular significancein understanding the interactions between PT and receptor molecules ontarget cells. FIG. 2 demonstrates that S1 sits on top of the planararrangement of the B-oligomer along its 5-fold central axis.

The two copies of S4 have essentially identical folds, and can besuperimposed with an rms difference for all 110 Cα atoms of 0.56 Å (0.32Å after omitting 21 Cα atoms in loops and at the C-terminus).Superposition of S2 and S3 shows a significant shift only at theC-terminus, with an rms difference for the remaining 188 Cα atom pairs(residues 4-91) of 0.63 Å. Secondary structure was assigned with DSSP(ref. 46). S1; 01:6-11, α1:15-21, β2:23-24, α2:32-37, β3:50-54,α3:57-77, β4:83-92, β5:97-99, α4:100-111, α5:118-127, β6:129-133,β7:135-136, β8:141-150, β9:155-162, β10:191-193, β11:198-199,α6:200-205, β12:225-227, α7:228-231. S2 and S3 N-terminal domains;β1:27-29 α1:32-37, α2:39-48, β2:54-56, β3:61-63, β4:70-72, β5:84-93. S2and S3 C-terminal domains, β6:100-105, β7:106-113, β8:119-125,09:128-135, α3:146-159, β10:163-173, 311:183-191. S4; β1:6-10, β2:11-20,β3:27-36, β4:48-55, α1:63-74, β5:78-89, β6:92-102. S5; β1:5-9, β2:10-20,β3:23-31, β4:37-43, α1:51-66, β5:70-74, β6:84-91.

Subunit S1

The N-terminal 175 residues of S1 of PT show structural homology withthe enzymatic portion of the A-subunit of LT, consistent with theirsimilar catalytic functions. All secondary structure elements withinthis region are conserved, except for helix α5 in PT which has noequivalent in LT. Strands β4 and β8, and helices α3 and α4 of PT arelonger than in LT, and there are significant differences in connectingloops. The disulphide bond in S1 (Cys-41/Cys-201) is not structurallyequivalent to the disulphide bond of LT, but in both cases reduction isessential for catalytic activity. Residues 176-235 of S1 have nostructural homology in LT. Analysis of C-terminal deletion mutants hasshown that this part of S1 is not essential for catalytic activity invitro (refs. 47,48). A stretch of residues in extended conformation isfollowed by two short strands (β10 and β11), a helix (α6), and a furtherstrand (β12). β-strands 10-12 form a small anti-parallel sheet. TheC-terminus enters a "pore" in the centre of the B-oligomer with a shorthelix (α7) and ends within a pore.

The amount of surface buried between S1 and B-oligomer is much largerthan between the A and B portions of LT. However, the interface in PT istypical of that found in stable oligomeric proteins (ref. 49). Thus, thenumbers of hydrophobic, hydrogen-bond and electrostatic interactions arenot unusual for protein interfaces of this size. The interaction betweenthe C-terminus of S1 (228 to 235) and the B-oligomer pore accounts for28% of the buried surface. Residues 211-220 of S1 (between α6 and β12)have poor density and could not be modelled. A disordered region occursin a roughly equivalent part of the LT A-subunit, at the junction of A1and A2, and proteolytic cleavage in this region is necessary formembrane translocation. Tryptic cleavage of S1 at Arg-218 has been foundto enhance activation of PT in vitro (ref. 50).

Active site

The structure of the active site of S1 is depicted in FIG. 3. A numberof amino acid residues previously found to be implicated in the activesite of S1 are included in this figure and labelled R9 (Arg-9), R13(Arg-13), W26 (Trp-26), H35 (His-35), C41 (Cys-41), S52 (Ser-52), E129(Glu-129) and C201 (Cys-201).

As expected, the active site of S1 is structurally homologous to theactive sites of LT, ETA and DT. FIG. 4 shows a sequence alignment ofstructurally equivalent residues in the active sites of these fourtoxins. Identical residues are high-lighted in bold lettering; only tworesidues, corresponding to Tyr-8 and Glu-129 in PT, are conserved in allfour toxins. These glutamate residues have also been shown to beessential for enzymatic activity in each of the toxins. In PT, the sidechain of Glu-129 is within hydrogen bonding distance of His-35, Ser-52and Gln-127. Mutation of His-35 or Ser-52 is also associated withconsiderably reduced ADP-ribosyltransferase activity (ref s. 19,64).Reduction of the disulphide bridge between Cys-41 and Cys-201 of S1 thatis required for expression of enzymatic activity may induce aconformational change permitting productive binding of NAD.

B-oliaomer

The C-terminal half of S2 and S3, both copies of S4, and S5 have similarfolds, consisting of six antiparallel β-strands forming a closedβ-barrel, capped by an α-helix between the fourth and fifth strands(FIG. 1). This fold has been recognized previously in several proteinswhich bind either an oligosaccharide or an oligonucleotide, and namedthe "OB fold" (ref. 51). Other proteins containing the OB fold includethe B-subunits of LT and VT-1. A sequence alignment based upon astructural superposition of the B-subunits of PT, LT and verotoxin-1 isshown in FIG. 5 and demonstrates that there is no detectable sequencehomology. Adjacent B-subunits associate mainly through antiparallelβ-sheet interactions to form a rather asymmetrical pentamer around acentral pore lined by five helices. The same type of B-subunitinteraction, but with more perfect 5-fold symmetry, is seen in theB-pentamers of LT and verotoxin-1. In PT, the most extensiveinteractions between B-subunit monomers are those of S2 with S4 and S3with S4, in agreement with the observation that the B-oligomerdissociates in 5 M urea into an S2/S4 dimer, an S3/S4 dimer and an S5monomer (ref. 52). The N-terminal half of S2 and S3 form separatedomains at the periphery of the B-oligomer. Each of these domainsconsists of five β-strands and two α-helices, with a fold similar tothat of the lectin domain from a rat mannose binding protein (MBP) (53)(FIG. 6), which belongs to a family of calcium dependent (C-type)eukaryotic lectins.

Receptor-binding sites

Most of the sequence differences between S2 and S3 are found near theN-termini, suggesting that these regions may contain determinants ofcarbohydrate binding specificity. The crystal structure shows that,although the N-terminal domains of S2 and S3 show overall structuralhomology with a member of the family of C-type eukaryotic lectins (FIG.6), they apparently lack its functional region. Thirty-two conservedamino acids in the sequences of known C-type lectins are believed tocontribute to the carbohydrate recognition domain (CaRD). Features ofthe N-terminal sequences of S2 and S3 thought to be characteristic ofthe CaRD motif were reported (ref. 8), but with the exception of thedisulphide bond (Cys-23/Cys-87), they are not consistent with theobserved structural relationship. None of the remaining CaRD residuescan be identified in the structure-based sequence alignment of S2 and S3with MBP (FIG. 7). In the MBP structure, the calcium ligands lie withina long loop between strands β2 and β3 that was found to be thecarbohydrate binding site (ref. 54). This loop has no structuralequivalent in S2/S3 (FIG. 6), and there is no evidence that calcium isrequired for receptor binding of PT.

Evaluation of experimental findings in the light of the crystalstructure points to two regions of the N-terminal domains of S2 and S3that may be involved in receptor binding. Both sites are exposed and lieon the probable membrane binding surface of the toxin (furthest fromS1). The first site is the loop between residues 18 and 23. Thissequence in S2 was found to be homologous to residues 62 to 67 in wheatgerm agglutinin (ref. 55), which form part of the sialic acid bindingsite determined crystallographically (ref. 56). Synthetic peptidesincorporating this sequence bind to sialic acid-containingglycoconjugates (ref. 55). These segments of the two crystal structuresare in fact very similar; residues 18 to 23 of S2 can be superimposed onresidues 62 to 67 of wheat germ agglutinin with an rms difference ofonly 1.03 Å for 22 main chain atoms. A second site that may have a rolein carbohydrate recognition is helix α2 (residues 39 to 48). Exchangemutation of residues 37 to 52 between S2 and S3 was reported to beassociated with an exchange of binding specificities (ref. 8).

The central pentameric domain of the PT B-oligomer may also be involvedin receptor binding, as in the previously recognized proteins thatcontain the OB fold. Crystallographically determined binding sites ofother proteins with this fold (ref. 51) are in variable loops betweenβ2-β3, β4-α and β5-β6 (VT-1 numbering), but they lie in similarlocations with respect to the overall structure. An extended bindingsite on S2 or S3 might span one or more of these C-terminal domain loops(β7-β8, β9-α3 and β10-β11), as well as parts of the N-terminal domain.Mutations in the vicinity of β5-β6 and β10-β11 of S2 and S3 have beenfound to affect receptor binding (refs. 21,22).

Another feature of the PT crystal structure relevant to cell surfacebinding activity is the observation that PT crystallizes as holotoxindimers. Association is mediated by edge-on interaction between pairs ofS2 and S4 subunits on each molecule, such that the two 5-fold axes arealmost parallel and the two cell-binding B-oligomer faces almostcoplanar. This arrangement is illustrated in FIG. 8, in which thepolypeptide chains are represented as line segments joining amino acidCα atoms. The area of surface buried at the interface is sufficient forstable complex formation in solution, suggesting that dimeric PTpresenting an extended multivalent cell-binding region is importantunder physiological conditions.

Example 4

This Example describes the identification of functional amino acidresidues in pertussis toxin by examination of its 3D structure.

Inspection of the crystal structure of PT in comparison with those ofLT, ETA and DT has permitted a molecular model of the active site of S1to be constructed (FIG. 3). This model accounts for the functionalimportance of a number of amino acid residues previously implicated inthe active site of S1 by the methods outlined above. It further providesdirection for the identification of additional previously unrecognizedresidues whose modification might be expected to yield PT analogs withaltered biological activity. Thus, in one embodiment of the presentinvention, there are provided amino acid residues in subunit S1 of PTcontributing to catalytic activity or binding of substrates oreffectors, which include nicotine adenine dinucleotide (NAD).

A model for NAD binding to S1 was generated by superimposing the activesite of PT onto DT in crystals of DT bound to a dinucleotideadenylyl-3',5'-uridine monophosphate (ref. 23). The position of theadenine moiety of this molecule, denoted by `Ad` in FIG. 3, mayrepresent the position of the adenine moiety of NAD bound to S1. Most ofthe amino acid residues already implicated in the catalytic activity ofS1 are clustered in a region adjacent to this site and are labelled inFIG. 3. They include R9 (Arg-9), R13 (Arg-13), W26 (Trp-26), H35(His-35), C41 (Cys-41), S52 (Ser-52), E129 (Glu-129) and C201 (Cys-201).

Also marked in FIG. 3 are several other amino acid residues located inspatially proximate relation to the previously identified amino acidresidues or with the predicted position of bound NAD, and therefore alsolikely to be important to the enzymatic activity of S1. These amino acidresidues are F23 (Phe-23), S48 (Ser-48), V51 (Val-51), Q127 (Gln-127),L131 (Leu-131), G199 (Gly-199) and A200 (Ala-200). Analogues of PT inwhich one or more of these amino acid residues are deleted, replaced ormodified are therefore predicted to exhibit altered biological activity.

A second approach to modifying PT is to interfere with its ability tobind to target cells. Another embodiment of this invention thereforerelates to the provision of amino acid residues in the B subunits of PTinvolved in or influencing binding to cell surface glycoconjugatereceptors.

The crystal structure points to the significance of an α-helical regionbetween residues 38 and 50 of subunits S2 and S3 in attachment of PT toglycoconjugate receptors. This region, seen at the base of therepresentation in FIG. 6, is also conserved in MBP according tostructure, and to some extent also according to sequence (FIG. 7). TheseS2 and S3 helices are also partially exposed and could contribute to acell-binding surface. It has been reported that sequence changes in thisregion of S2, or exchange of sequences between S2 and S3, affect thecarbohydrate binding specificity of isolated S2 and S3 subunits (ref.8). Examination of the crystal structure of PT shows that this helixforms one face of a groove across the surface of S2 and S3 into whichthe most functionally sensitive amino acid residues are directed. Theopposite face of this groove is occupied by amino acid residues His-15,Gln-16, Leu-82 and Lys-83 in S2, and by residues Gln-15, Gln-16, Tyr-82and Arg-83 in S3. Replacement of Tyr-82 of S3 has already been shown todiminish the biological activity of PT (ref. 22), but the significanceof the other sites to glycoconjugate binding of PT has not previouslybeen recognized. Analogues of PT in which one or more of these aminoacid residues are deleted, replaced or modified are therefore predictedto exhibit altered biological activity.

Dimerization of PT might also be expected to affect its ability to bindto target cells. Although dimerization occurs between pairs of S2 and S4subunits on two adjacent PT molecules, similar dimerization betweenpairs of S3 and S4 subunits is stereochemically possible but does notoccur, indicating that S2 contains unique amino acid determinantspromoting dimerization. A comparison between S2 residues at the dimerinterface and the corresponding S3 residues is as follows:

    ______________________________________                                        Residue No.                                                                             20  52  54  63  66  67  81  82  83                                  ______________________________________                                        Subunit S2                                                                              Tyr Trp Ile Leu Glu Tyr Asp Leu Lys                                 Subunit S3                                                                              Tyr Trp Ile Leu Ala Tyr Ile Tyr Arg                                 ______________________________________                                    

The S2 residues Glu-66, Asp-81, Leu-82 and Lys-83 not conserved in S3are therefore predicted to be responsible for dimerization of PT. Thusanalogues of PT in which one or more of these amino acid residues aredeleted, replaced or modified are expected to exhibit altered biologicalactivity. As described above, amino acid residues 82 and 83 were alsoimplicated in glycoconjugate binding on other grounds.

Detailed examination of the crystal structure of PT reveals thatsignificant interactions between S2 and S4 subunits on adjacentmolecules also involve residue Trp-52 of S2 and residues Asp-1, Tyr-4,Thr-88 and Pro-93 of S4. Analogues of PT in which one or more of theseamino acid residues are deleted, replaced or modified are therefore alsopredicted to exhibit altered biological activity.

A third approach to detoxification of PT is to impede the ability of thecatalytic S1 subunit to dissociate from the B-oligomer, which isimportant in the context of in vitro enzymatic activity, and is believedalso to be important in vivo. A further embodiment of this inventiontherefore relates to the identification of amino acid residues ofsubunit S1 likely to influence the ability of a functional moiety of S1to disengage from the remainder of the molecule.

A significant feature of subunit S1 as it appears in the crystalstructure is the lack of electron density associated with amino acidresidues 211-220, indicating that this portion of the polypeptide chainis disordered and unlikely to be essential for polypeptide chain foldingor stabilization. The amino acid sequence corresponding to this regionis as follows:

    Residue No.  211             215                 220                          Sequence    Ala Met Ala Ala Trp Ser Glu Arg Ala Gly  (SEQ ID NO: 45)      

However, the downstream C-terminal sequence 228 to 235 is observed to beimportant in establishing contact between S1 and the central pore of theB-oligomer alluded to previously. A similar situation exists in thecrystal structure of LT, where a disordered region occurs in astructurally analogous part of the catalytic LT-A subunit just upstreamof the C-terminal segment which interacts strongly with the LT-B pore.Significantly, proteolytic cleavage in this disordered region isnecessary for activation of LT. It has not been previously suggestedthat proteolytic cleavage of S1 is required for activation of PT;however, the S1 segment 211 to 220 contains recognition sites fortrypsin (Arg-217) and chymotrypsin (Met-212 and Trp-215), and is readilycleaved by these enzymes in vitro (ref. 50). Moreover, a truncated formof S1 lacking all amino acid residues beyond position 180 is known toexhibit almost full NAD-glycohydrolase activity in vitro in the absenceof B-oligomer (ref. 57), indicating that the C-terminal region is notessential once S1 is disconnected from the rest of the PT molecule.Deletion or truncation of the loop between S1 residues 211 and 220, orsubstitution of key amino acid residues within this loop, may thereforeyield PT analogs with altered biological activity.

Upstream of the region of disordered structure in S1 is a hydrophobicregion extending from residues 184 to 203, which has the characteristicspredicted for a membrane-spanning segment (ref. 58). The amino acidsequence corresponding to this region is as follows:

    Residue No.  184                     190                                      Sequence     Val Ala Ser Ile Val Gly Thr Leu Val Arg  (SEQ ID NO: 46)         Residue No.      195                 200         203                          Sequence     Met Ala Pro Val Ile Gly Ala Cys Met Ala                      

Reduction of the disulfide bond between Cys-41 and Cys-201, togetherwith proteolytic cleavage of the C-terminus as described above, aretherefore predicted to expose a region of S1 suited to anchoring afunctional moiety of S1 in a cellular membrane. This functionality ispredicted to facilitate translocation of S1 and its diffusion along anintracellular membrane. Introduction of polar amino acid residues withinthis region may therefore yield PT analogues with altered biologicalactivity.

Example 5

This Example describes the generation of a crystalline complex betweenPT and a biantennary undecasaccharide derived from human serumtransferrin, and determination of its 3D structure. The composition ofthis carbohydrate, obtained from BioCarb Chemicals, is as follows:##STR1##

PT was purified and crystallized as described in Example 1. Nativecrystals were soaked in a 10 mM solution of the carbohydrate in 25 mMNa/K-phosphate, 0.3 M KCl, 0.02% NaN₃, pH 8.0 for 5 h, or until thesurface became etched, then removed for mounting. Crystals dissolvedwhen soaked in a 30 mM solution of the carbohydrate. Data were collectedfrom a single crystal using a synchrotron radiation source and imageplate detector on the Weissenberg camera (ref. 34). The data wereprocessed using WEIS (ref. 35) and programs in the Daresbury CCP4package, and were 87% complete between 10 and 3.5 Å, with a mergingR-factor of 9.7% (25.1% at 3.5 Å) and multiplicity of 3.4.

Crystals of the PT-sugar complex remained isomorphous with the native PTcrystals (space group P212121, with cell dimensions a=163.8 Å, b=98.2 Å,c=194.5 Å). As before, the asymmetric unit contained two moleculesrelated by non-crystallographic two-fold symmetry. However, the PT-sugardata merged poorly with data from native crystals (R_(merge) onamplitudes 23.5%). The structure was solved by rigid body refinement ofthe native PT structure against the PT-sugar data using X-PLOR (ref.42). Initially each toxin molecule in the asymmetric unit was refinedindependently (two rigid groups), and later the two molecules weredivided into their six individual subunits (12 rigid groups). Thestarting R-factor was 36.5% (10-8 Å), and fell to 22.0% (10-4 Å) at theend of rigid-body refinement. The resulting model showed an overallrotation of 1.2° relative to the original native PT structure, but therewere no significant internal domain shifts.

A difference electron density map was calculated using the new modelphases with SIGMAA-weighted Fourier coefficients (ref. 43). The threehighest peaks per asymmetric unit on the map (7.0-8.4 times the rmselectron density) corresponded to three sugar moieties, which weremodelled using the program O (ref. 41). Although bound carbohydrate wasobserved at equivalent sites on subunits S2 and S3, only three of thefour possible binding sites in the asymmetric unit were occupied. Thefourth potential site, on the S2 subunit of molecule 2, was evidentlynot accessible to this carbohydrate owing to a packing interaction witha symmetry-related PT molecule. Crystals dissolved when soaked in 30 mMcarbohydrate, perhaps because this interaction was disrupted.

At each occupied binding site, clear electron density was present foronly two carbohydrate residues, the other nine remaining disordered. Thechemical identities of the ordered residues were assigned by assessingthe fit of every possible pair of neighbouring residues in thetransferrin undecasaccharide to the electron density of a differenceFourier map. Structure factors and phases for this map were calculatedusing the model after rigid body refinement (Example 5), and before anyattempt had been made to build the carbohydrate. Part of this map isshown in FIG. 9. Although the resolution of the diffraction data is only3.5 Å, the result was unambiguous: only the terminal NeuAc(2α,6)-Galmoiety could fit the observed electron density. The linkages between allother adjacent pairs of sugar residues were such that both residuescould not be placed in density at the same time.

Two additional amino acid residues at the N-terminus of S2 of PTmolecule 2 were added to the native PT model, and the complete structurewas refined by simulated annealing with X-PLOR, using an additionalempirical energy function for carbohydrate (ref. 59). The conformationof the bound NeuAc(2α,6)-Gal group was found to be almost identical ateach binding site, and close to one of the low energy conformations forthis molecule determined using energy calculations and NMR spectroscopy(ref. 60).

The current PT/sugar model contains 1870 amino acid residues (934 inmolecule 1 and 936 in molecule 2), six carbohydrate residues, and nosolvent. Atomic temperature factors are those of the native PT model,except for unique atoms (including the carbohydrate) which have fixedB-factors of 20 Å². The crystallographic R-factor is 18.3% for allreflections between 10.0 and 3.5 Å. The rms deviation from ideality is0.015 Å for bond distances and 1.9° for bond angles. The rms differencein coordinates of all main chain atoms after superposition of the twomolecules is 0.18 Å.

Example 6

This Example describes the analysis of the structure of the complexbetween pertussis toxin and the undecasaccharide from human serumtransferrin, and the identification of functional amino acid residues inpertussis toxin.

The binding sites of the disaccharide moieties on S2 and S3 in relationto the complete structure of PT are shown in FIGS. 10 and 11. Theshortest distance between binding sites in the asymmetric unit is ˜65 Å,which excludes the possibility of bivalent interaction of a single sugarMolecule through its two equivalent arms. Interactions between thecarbohydrate and the protein are virtually identical at each of thethree binding sites in the asymmetric unit. The galactose makes nointeractions with the protein, but the sialic acid is within hydrogenbonding distance of polar or charged groups on Tyr-102, Ser-104 andArg-125, as detailed in Table 4 below and depicted in FIG. 12. Inaddition, the sialic acid ring makes hydrophobic contacts with thearomatic rings of Tyr-102 and Tyr-103. All amino acid residues thatinteract directly with the sialic acid are fully conserved between S2and S3.

The present finding of three sialic acid binding sites per asymmetricunit agrees with a recent report (ref. 61) describing an isomorphousderivative of the same PT crystal form. In that case, native PT crystalswere soaked in a mercuric derivative of sialic acid, resulting in threemercury sites, as identified by difference Patterson techniques. Thepositions of peaks in the published Harker sections are consistent withthe mercury atoms lying within 1 to 2 Å of the position of C6 ofgalactose in the undecasaccharide complex. This suggests that thebinding mode for sialic acid is very similar in the two cases.

The transferrin undecasaccharide used in this study was chosen becauseits composition and linear structure are typical of the oligosaccharidechains of glycoproteins, such as fetuin, known to bind strongly to PT,and of the surface glycoproteins of mammalian cells, such as Chinesehamster ovary cells, that are susceptible to intoxication by PT.Furthermore, the crystallographic results described herein areconsistent with the findings that asialofetuin has severely reducedaffinity for PT (ref. 6), while a variant CHO cell line lacking terminalsialic acid residues does not bind PT and is not susceptible to itsaction (ref. 7). These observations indicate that the terminal sialicacid residues of glycosyl chains are important for the physiologicallysignificant binding of PT to mammalian cells, and suggest that thebinding site on PT disclosed by the present invention has physiologicalsignificance.

The observed sugar interaction site lies in the C-terminal portion ofS2/S3, which has the oligomer-binding fold (ref. 51) also present in thecell-binding B-subunits of members of the cholera toxin and Shiga toxinfamilies. This portion of the structure is thus expected to contributeto binding to cell surface receptors, although additional sites are alsolikely to exist in the N-terminal domains of S2 and S3, as discussedabove. The S2 and S3 residues observed to interact with the sugar areindeed consistent with some experimental studies of receptor binding.There is good evidence that tyrosines are involved; firstly, it has beenfound that the toxin is inactivated by iodination (which typicallyaffects tyrosines), but can be protected by binding to fetuin-agarose(ref. 62); secondly, recombinant forms of PT with mutations or deletionsinvolving Tyr-102 and Tyr-103 of S2 or S3 show reduced biologicalactivities (refs. 21,22). Cell-binding activity is also attenuated bydeletion of Asn-105 in S2 or Lys-105 in S3 (refs. 21,22), residues thatare in close proximity to those observed in the crystal structure tointeract direct with the sugar. However, the central importance ofresidues Ser-104 and Arg-125 of S2 and S3 in binding sialic acidresidues has not been previously recognized. Analogues of PT in whichone or more of these amino acid residues are deleted, replaced ormodified are therefore predicted to exhibit altered biological activity.

Example 7

This Example describes the generation of a crystalline complex betweenPT and ATP, and determination of its 3D structure.

PT was purified and crystallized as described in Example 1. Nativecrystals were soaked in a 30 mM solution of ATP in 25 mM Na/K-phosphate,0.3 M KCl, 0.02% NaN₃, pH 8.0 for 1.5 day. Data were collected from asingle crystal using a synchrotron radiation source and image platedetector on the Weissenberg camera (ref. 34). he data were processedusing WEIS (ref. 35) and programs from the BIOMOL package (obtained fromthe laboratory of Chemical Physics, University of Groningen, Nijenborgha16, 9747 AG, Groningen, Holland), and were 57% complete to 2.5 Å, with amerging R-factor of 9.3% (33.1 at 2.5 Å) and a multiplicity of 3.0.Owing to radiation damage during data collection, the maximum resolutionof the diffraction decreased from 2.5 Å for the first images to 3.3 Åfor the last. Therefore data completeness beyond 3.3 Å dropped ratherquickly to 18% for the highest resolution shell. At 3.3 Å the data were88% complete, with a merging R-factor of 7.6% (17.5% at 3.3 Å) and amultiplicity of 3.3.

Crystals of the PT-ATP complex remained isomorphous with the native PTcrystals (space group P2₁ 2₁ 2₁, with cell dimensions a=163.8 Å, b=98.2Å, c=194.5 Å). As previously, the asymmetric unit contained twoholotoxin molecules related by non-crystallographic two-fold symmetry,and the data merged reasonably well with the native data (R_(merge) onamplitudes was 15.4% for all reflections in the range 30.0-2.9 Å). Thestructure was solved by rigid body refinement of the native PT structureagainst the PT-ATP data, using X-PLOR (ref. 42). The B-values for allatoms were reset to 25 Å², and all 12 subunits were treated asindependent rigid bodies. The starting R-factor was 31.1% (10-3 Å) andfell to 28.4% (10-3 Å).

A difference electron density map was calculated using the new modelphases with SIGMAA-weighted Fourier coefficients (ref. 43). In theresulting map, the density for the ATP molecule was clearly recognizableas the most prominent feature of the map. Using the program O (ref. 41),a model of the ATP molecule was inserted into the difference electrondensity. The density also indicated that the C-terminus of subunit S1and the side chain of Arg-69 of one of the S4 subunits, denoted S4b,changed their conformation. To obtain an unbiased density for theseatoms, the C-terminal residues 234 and 235 of subunit S1 were removedand Arg-69 of S4b was modified to an alanine residue. Positionalrefinement of this model, with a restraint to keep the twonon-crystallographically related holotoxin molecules in the asymmetricunit similar, decreased the R-factor from 29.2% (8.0-2.7 Å) to 25.5%(8.0-2.7 Å). In a new difference map, calculated as described aboveusing the phases of the new model, there was clear electron density forthe two C-terminal residues of subunit S1 and the Arg-69 side chain,which were reintroduced into the model.

Repeated cycles of model building and X-PLOR refinement finally resultedin a model for the complex in which each member of the dimer contained935 amino acid residues (7259 atoms) and 31 atoms from the bound ATPmolecule. In addition 81 water molecules were added per asymmetric unit.The R-factor for this model was 22.9% (8.0-2.7 Å), with tightlyrestrained B-values (rms difference between bonded atoms 1.3 Å²) andprotein geometry (rms deviations from ideal bond lengths and angles0.017 Å and 2.0° respectively).

Example 8

This Example describes the analysis of the structure of the complexbetween pertussis toxin and ATP, and the identification of functionallyimportant amino acid residues in pertussis toxin.

The binding site of ATP in PT molecule 1 is shown in FIGS. 13 to 15.FIGS. 13 and 14 demonstrate that the ATP molecule is located in thecentral pore created by the pentameric B-oligomer, very close to theC-terminus of subunit S1. FIG. 15 illustrates amino acid residues thatsignficantly interact with the ATP molecule. For simplicity, theindividual subunits S1, S2, S3, S4a, S4b and S5 are denoted A, B, C, D,E and F, respectively. The ATP binding site in molecule 2 is almostidentical. list of all protein atoms within 3.5 Å of at least one ATPatom in molecule 1 is given in Table 5 below.

The binding of ATP to the holotoxin does not cause global changes in thequaternary or tertiary structure. The most prominent changes aredisplacements of the C-terminal three residues of the S1 subunit and theside-chain atoms of Arg-69 of subunit S4b, which are required in orderto accommodate the ATP molecule. In addition, movement of the S1 tailhelps to reduce the unfavourable electrostatic interactions between thenegatively charged phosphate groups of ATP and the negative C-terminalcarboxylate group of S1, while movement of Arg-69 enhances thefavourable electrostatic interactions between the phosphates and thepositive side chain.

Binding of ATP

The triphosphate moiety of the ATP molecule is surrounded by aremarkable number of positively charged amino acid side chains. ThusArg-150 and Lys-151 of S2, Arg-150 and Arg-151 of S3, and Arg-69 of S4bare all within 3.2 Å of at least one phosphate oxygen. The C-terminalcarboxylate group of subunit S1 is the only protein-derived negativecharge within this range of a phosphate oxygen atom. Accordingly, it canbe expected that the electrostatic interactions between the threenegative charges on the phosphates and the surrounding positive chargesof the protein contribute significantly to the overall binding energy ofATP. This conclusion is consistent with studies that show that bindingof ADP to pertussis toxin is considerably weaker than that of ATP (refs.3,4).

The phosphate oxygen atoms O3, O5, O6, O7, O8, O9 and O10 all makehydrogen bonds to at least one protein atom. Important protein residuesinvolved in these hydrogen bonds are the above-mentioned Arg-150 andLys-151 of S2, Arg-150 and Arg-151 of S3, and Arg-69 of S4b, plus Ser-61of S4b. Oxygens O2 and O4 appear not to make hydrogen bonds to theprotein. The oxygen atoms O2' and O3' of the ribose unit of ATP formhydrogen bonds to Ser-61, Glu-65 and Arg-69 of subunit S4b. Of the fournitrogen atoms in the purine ring of ATP, only N7 takes part in ahydrogen bond, to residue Ser-62 of subunit S5. The other three do nothave any contacts within 3.5 Å. However, extensive Van der Waalsinteractions exist between the purine ring and the alkyl side chain ofArg-69 of S4b. In addition, the phenyl ring of Phe-59 of S5 plays animportant role in shaping the cavity for the purine ring, even thoughits closest distance to the purine ring is 4.4 Å (this residue notlisted in Table 5).

Another aspect of the binding of ATP to PT relates to the fact that ATPoccupies a site in the central pore of the B-oligomer which wouldnormally be filled with solvent molecules. Most if not all of thesesolvent molecules would be expected to have some degree of structure, inview of the concentration of charged amino acid residues in thevicinity. Release of these water molecules would increase their entropy,thus giving a favourable contribution to the binding energy of the ATPmolecule.

Functional amino acid residues

The functional importance of ATP binding to pertussis toxin lies in itsability to loosen the interaction between the catalytic S1 subunit andthe B-oligomer, or allow its complete release (refs. 4,67). For example,in the absence of ATP, the in vitro ADP-ribosyltransferase activity ofPT is reduced by 20-fold (ref. 67). Modifications to the structure of PTthat prevent the release of subunit S1 can thus be expected tosubstantially affect the biological activity of PT. Accordingly,modifications that prevent ATP binding would also be expected toattenuate the biological activity of PT. Amino acid residues of PT shownto participate in binding ATP according to the forgoing crystallographicanalysis are Arg-150 and Lys-151 of subunit S2, Arg-150 and Arg-151 ofsubunit S3, Ser-61, Glu-65 and Arg-69 of subunit S4b, and Ser-62 ofsubunit S5. Of these, Arg-69 of subunit S4b is particularly important inview of its involvement in both polar and non-polar interactions withATP. Ser-61 of subunit S4b and Ser-62 of S5 are significant as being thesole hydrogen bonding partners of O5 and N7 of ATP, respectively, aswell as lining the the binding pocket for the ATP molecule. In addition,Gly-58 of S5 occupies a position where any larger amino acid residuewould cause steric hindrance of a bound ATP molecule. Analogs of PT inwhich one or more of these amino acid residues are deleted, replaced ormodified are therefore predicted to exhibit altered biological activity.

An alternative approach to hindering the release of S1 required foractivation of the toxin is to modify the C-terminus of S1 itself, sincethis segment is pushed aside on binding ATP. Thus extension of the S1chain by one amino acid would be expected to impede the approach of anATP molecule. Addition of a negatively-charged amino acid or replacementof the terminal Phe-235 with a negatively charged amino acid residuewould provide additional electrostatic repulsion with respect to thetriphosphate tail of ATP, while augmenting favourable electrostaticinteractions with neigbouring positively charged residues of the Bsubunits. Such changes would therefore be expected to yield PT analogswith altered biological activity.

Entrance pathway of ATP

To reach its binding site, the ATP molecule must approach the holotoxinfrom the side of the B-oligomer remote from S1, since no entrancepathway can be found from the opposite side which is blocked by the S1subunit. Blocking the entrance to this binding site would be expected toprevent activation of the toxin. Amino acid residues flanking theapproach to the binding site are Ser-147 of S2, Gly-60 and Ser-61 ofsubunits S4a and S4b, and Asp-54, Thr-51 and Thr-55 of S5. Modificationsto these residues in order to alter their size or polarity, therefore,would be expected to diminish the ability of ATP to bind to PT and henceits ability to activate the holotoxin.

Example 9

This Example describes the production of PT holotoxin analoguescontaining mutations at the specific sites identified by examination ofthe crystal structures disclosed herein.

To produce PT analogues containing site-specific mutations, the toxoperon may be mutated and the mutant tox operon expressed in recombinantstrains of Bordetella parapertussis or Bordetella pertussis. The extentof detoxification is then estimated by an ADP-ribosyltransferase assay,by the CHO cell clustering assay, and by in vivo assays includinghistamine sensitisation and lymphocytosis-promotion. The techniquesrequired to produce and analyze these analogues are reported in thefollowing references, each of which is incorporated herein by reference:

U.S. Pat. No. 5,085,862

U.S. Pat. No. 5,221,618

Loosmore, S. et al. (ref. 15)

Zealey, G. et al. (ref. 65)

Zealey, G. et al. (ref. 66)

Loosmore, S. et al. (ref. 22)

Briefly, DNA restriction enzymes were purchased from Bethesda ResearchLaboratories, Boehringer Mannheim, New England BioLabs, and Pharmaciaand used according to the specifications of the manufacturers, Allradioisotopes were supplied by New England Nuclear. Standard recombinantDNA techniques were performed as described by Maniatis et al.

The tox operon from the Connaught vaccine strain 10536 and its flankingregions were cloned from a Charon 35 phage library into pUC plasmids.The S1 gene was subcloned into M13mp18 for in vitro site-directedmutagenesis by the phosphorothioate procedure (Amersham). all mutationswere confirmed by the dideoxy sequencing method of Sanger et al. Mutatedtox operons were cloned into the replicating plasmid pRK404 (ref. 68)and introduced into B. parapertusis by conjugation for production of PTanalogs.

B. pertussis strains were maintained in Bordet-Gengou medium containing20% fresh defibrinated sheep blood and cultured in Stainer-Scholtemedium supplemented with 0.2% (2,6-O-dimethyl)β-cyclodextrin. Culturesupernatants were typically harvested after 3 days of growth at 36° C.

Enzyme immunoassays. PT concentration was determined by fetuin captureenzyme-linked immunosorbent assay (ELISA). Nunc Immulon II ELISA plateswere coated with fetuin (GIBCO; 2 μg/ml) for 16 h at 4° C. in 50 mMsodium carbonate, pH 9.6. The plates were then washed three times inphosphate-buffered saline (PBS) containing 0.05% Tween 20. Wild-type PTor sample PT was serially diluted and added to the wells, and the plateswere incubated for 30 min at room temperature. After three washes, boundPT was detected with monospecific rabbit anti-PT or anti-S1 antibodiespurified by protein A affinity chromatography (Pharmacia) and conjugatedto peroxidase. The plates were washed, and tetramethyl benzidine(Allelix Diagnostics Inc.) in 0.005% hydrogen peroxide was added todetermine the amount of bound enzyme. The reaction was stopped by theaddition of 1 M H₂ SO₄. The A₄₅₀ was determined by using a referencewavelength of 540 nm (Biotek EIA Autoreader model EL310).

Antigenicity immunoassay. A panel of murine monoclonal anti-PTantibodies was developed in our laboratories from splenocytes of miceimmunized with detoxified PT. The monoclonal antibody PS21 was shown torecognize the S1 subunit in association with free and fetuin-bound Boligomer. The LP12 monoclonal antibody recognizes the S3/S4 dimer.Polyclonal anti-PT antisera were raised in rabbits immunized withpurified PT.

The analogs of PT may have one or more of these amino acid residuesdeleted, replaced or modified either singly or severally, and either inthe absence of or in addition to other mutations. The amino acidresidues described herein could be replaced by any other α-amino acid,for example, but not exclusively, by means of genetic manipulation in acellular expression system or by chemical synthesis of individualsubunits. It is also understood that certain of these amino acidresidues could also be modified subsequent to protein synthesis bychemical or biochemical methods. Such approaches are described in thescientific literature and will be familiar to those skilled in the art.

One consideration in the selection of amino acid residues with which toreplace the identified natural amino acid residues is the likelyfunctions of the natural amino acid residues in the toxin, and theirspatial and chemical characteristics in relation to the availablereplacements. This process is facilitated by the exclusive ability ofthe inventors to inspect the detailed three-dimensional structure of PToutlined in this document, and appraise the unique environment of eachamino acid residue.

The present invention, therefore, provides PT analogs in which the aminoacid residues specified according to subunit in Table 3 below aredeleted, replaced or modified alone or in combination with additionalmutations. Preferred amino acid replacements at each site are alsoshown.

The present invention also provides pertussis toxin analogues in whichat least one of the amino acid residues from position 211 to position220 of subunit S1 is deleted, substituted or modified, either exclusiveor inclusive of additional mutations in the PT molecule. Preferably, butnot exclusively, one goal of such sequence changes is to remove proteasecleavage sites signalled by Met-212, Trp-215 and Arg-217. Examples ofpreferred amino acid substitutions at these positions are as follows:

Met-211→Ser or Thr

Trp-215→Asn or His

Arg-217→Ser or Gln

The present invention further provides pertussis toxin analogues inwhich the hydrophobic region between residues 184 and 203 of subunit S1is rendered more hydrophilic by substitution of one or more non-polarresidues by polar or charged residues.

Referring to Table 6, there is shown the characterization of PT analogshaving mutations S2 Arg-125→Ala, S3 Arg-125→Ala and S2 Lysine-83→Ala. Todetermine the concentration of PT analog production, analogs werecaptured with a polyclonal anti-PT antiserum and detected with a furtherpolyclonal anti-PT antiserum. It is considered that this ELISA gives themost reliable measure of PT analog concentration, since neither capturenor detection depends upon maintenance of any particular single epitope.The ability of PT analogs to bind to fetuin was taken as a measure ofthe integrity of the fetuin binding sites on S2 and/or S3. Thus, theratio of the concentration of PT analog determined by the fetuin captureELISA and the polyclonal capture ELISA (corrected for the ratio of thisratio for wild-type PT) is a measure of the detoxification of the PTanalog.

The results shown in Table 6 indicate that PT analogs having amino acidreplacements at sites in the S2 and S3 subunits modified by a methodcomprising (a) identifying at least one amino acid residue of theholotoxin for modification by analyzing a three-dimensional structure ofcrystalline pertussis holotoxin determined by X-ray crystallography inrelation to known information concerning protein structure and function;(b) effecting mutagenesis of a tox operon encoding the holotoxin toremove or replace a nucleotide sequence coding for said at least oneamino acid residue and to produce a mutant tox operon; and (c)expressing the mutant tox operon in a Bordetella organism to produce themodified holotoxin, have altered biological activities. In thisembodiment the modified biological activity is cell binding.

It is emphasized that the described amino acid replacements onlyexamples of convenient replacements of the identified amino acidresidues to disrupt the function of the natural amino acid residues atthese locations. Other substitutions, modifications or deletions,according to the methods outlined above, may also be used to achievemodification of pertussis toxin, and are within the scope of the presentinvention.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides thethree-dimensional crystal structures of pertussis toxin and of complexesof PT bound to functionally relevant molecules, as determined by X-raycrystallography. These provide the ability to identify functional aminoacids for modification to alter the biological properties of PT.Modifications of such amino acids are possible within the scope of thisinvention.

                  TABLE 1                                                         ______________________________________                                        Oligosaccharides suitable for forming complexes                               with pertussis toxin for crystallographic analysis                            Name              Formula                                                     ______________________________________                                        lactose           Gal(1β,4)Glc                                           N-acetyllactosamine                                                                             Gal(1β,4)GlcNAc                                        3'-sialyllactose  Neu5Ac(2α,3)Gal(1β,4)Glc                         3'-sialyl-N-acetyllactosamine                                                                   Neu5Ac(2α,3)Gal(1β,4)GlcNAc                      6'-sialyllactose  Neu5Ac(2α,6)Gal(1β,4)Glc                         6'-sialyl-N-acetyllactosamine                                                                   Neu5Ac(2α,6)Gal(1β,4)GlcNAc                      lacto-N-biose     Gal(1β,3)GlcNAc                                        2'-fucosyllactose Fuc(1α,2)Gal(1β,4)Glc                            3'-fucosyllactose Gal(1β,4)[Fuc(1α,3)Glc]                          Lewis.sup.x trisaccharide                                                                       Gal(1β,4)[Fuc(1α,3)GlcNAc]                       Lewis.sup.a trisaccharide                                                                       Gal(1β,3)[Fuc(1α,4)GlcNAc]                       sialyl-Lewis.sup.x                                                                        Neu5Ac(2α,3)Gal(1β,4)[Fuc(1α,3)GlcNAc]           1β-2 N-acetylglucosamine-mannose                                                           GlcNAc(1β,2)Man                                        biantennary undecasaccharide                                                  Neu5Ac(2α,6)-Gal(1β,4)-GlcNAc(1β,2)-Man(1α,6)                      Man(1β,4)-GlcNAc(1β,4)-GlcNac                            Neu5Ac(2α,6)-Gal(1β,4)-GlcNAc(1β,2)-Man(1α,3)           ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Summary of pertussis toxin X-ray diffraction data                                       Native 1                                                                           Native 2                                                                           PIP  (NH.sub.3).sub.2 Pt(NO.sub.2).sub.2                                                   KlrCl.sub.6                                                                        OsCl.sub.3                                                                         KAuCN.sub.2                        __________________________________________________________________________    Number of crystals                                                                      1    2    1    1       1    1    1                                  Resolution (Å)                                                                      3.3  2.9  3.4  3.4     3.6  3.4  3.4                                Unique reflections                                                                      40277                                                                              52740                                                                              37389                                                                              37200   31494                                                                              31389                                                                              38182                              Completeness (%)                                                                        84   75   85   85      85   71   87                                 Multiplicity                                                                            3.5  6.1  3.4  3.5     3.4  2.9  3.5                                R.sub.merge (%).sup.a                                                         Overall   8.6  9.0  10.3 10.8    10.5 10.6 8.9                                Outer resolution shell                                                                  23.2 24.0 24.1 23.4    20.8 23.3 21.3                               R.sub.deriv (%).sup.b                                                                   --   --   15.9 15.2    24.3 20.4 11.8                               Number of sites                                                                         --   --   16   11      7    7    4                                  Phasing power.sup.c                                                           at 6.0 Å                                                                            --   --   1.71 1.33    0.96 0.88 0.46                               at 3.5 Å                                                                            --   --   1.19 0.88    0.79 0.62 0.26                               __________________________________________________________________________     PIP = diiodobis-(ethylenediamine) diplatinum(II)nitrate.                      .sup.a R.sub.merge = ΣΣ|l.sub.i -                        <|>|/ΣΣl.sub.i.                                 .sup.b R.sub.deriv = Σ||F.sub.PH | -         |F.sub.p ||/Σ|F.sub.p               |, where |F.sub.p | comes from native 2 data.      .sup.c Phasing power = |Σ|F.sub.H |.sup.     /Σ(|F.sub.PH (obs)| - |F.sub.PH              (calc)|).sup.2 |.sup.1/2.                              

                  TABLE 3                                                         ______________________________________                                        Functional amino acid residues in pertussis toxin                             Subunit   Residues   Preferred Replacement                                    ______________________________________                                        S1        Phe-23     Asp or Glu                                                         Ser-48     Ala                                                                Val-51     Ile                                                                Gln-127    Ala or Asp                                                         Leu-131    Lys or Arg                                                         Gly-199    Val or Gln                                                         Ala-200    Ile                                                                Phe-235    Glu                                                      S2        His-15     Ala or Thr                                                         Gln-16     Ala or Thr                                                         Trp-52     Val                                                                Glu-66     Ala or Lys                                                         Asp-81     Ala or Ser                                                         Leu-82     Ala or Glu                                                         Lys-83     Glu                                                                Ser-104    Ala                                                                Arg-125    Ala                                                                Ser-147    Thr                                                                Arg-150    Ser                                                                Lys-151    Ser                                                      S3        Gln-15     Ala or Thr                                                         Gln-16     Ala or Thr                                                         Tyr-82     Ala or Val                                                         Arg-83     Glu                                                                Ser-104    Ala                                                                Arg-125    Ala                                                                Arg-150    Ser                                                                Arg-151    Ser                                                      S4        Asp-1      Ala                                                                Tyr-4      Ala or Val                                                         Gly-60     Val                                                                Ser-61     Ala                                                                Glu-65     Ala                                                                Arg-69     Ala                                                                Thr-88     Val                                                                Pro-93     Ala                                                                Asp-54     Glu                                                                Thr-51     Tyr                                                                Thr-55     Tyr                                                                Gly-58     Val                                                      S5        Ser-62     Ala                                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Hydrogen bonding distances between the sialic acid moiety of                  the transferrin undecasaccharide and subunits S2 or S3 of                     PT at three equivalent binding sites in the PT-carbohydrate complex.                         Hydrogen-bond distance (Å)                                                at each binding site                                           sialic               S2 of     S3 of S3 of                                    acid atom                                                                              protein atom                                                                              Mol 1     Mol 1 Mol 2                                    ______________________________________                                        601A     OG of Ser-104                                                                             2.8       2.9   2.8                                      01B      N of Ser-104                                                                              3.1       2.7   2.8                                      08       NH2 of Arg-125                                                                            2.6       2.5   2.4                                      09       NH1 of Arg-125                                                                            2.6       2.5   2.5                                      N5       O of Tyr-102                                                                              3.0       2.7   2.6                                      ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Interaction distances less than 3.5 Å                                     between ATP and the protein in the PT-ATP complex                                                          ATP  Distance                                    Subunit   Residue   Atom     Atom (Å)                                     ______________________________________                                        S1        Phe-235   N        O3   3.20                                                  Phe-235   N        O3   3.20                                                  Phe-235   C        O10  3.41                                                  Phe-235   OT1      O10  3.02                                                  Phe-235   OT2      P1   2.99                                                                     O2   3.34                                                                     O3   2.74                                                                     O4   2.68                                                                     O7   3.25                                                                     P3   3.45                                                                     O8   3.31                                                                     O10  3.11                                        S2        Arg-150   NH2      O10  3.12                                                  Lys-151   CE       08   3.17                                                  Lys-151   NZ       O8   3.01                                        S3        Arg-150   NH2      O6   3.24                                                                     O7   3.35                                                  Arg-151   NH2      P3   3.04                                                                     O9   2.80                                                                     O10  2.43                                        S4b       Met-18    CE       O4'  3.27                                                  Ser-61    OG       O2'  3.07                                                                     P2   3.37                                                                     O5   2.93                                                                     O6   2.97                                                  Glu-65    CD       O3'  3.39                                                  Glu-65    OE1      C3'  3.07                                                                     O3'  2.50                                                  Arg-69    NE       N7   3.34                                                  Arg-69    CZ       N7   3.21                                                                     C8   3.12                                                                     O3   3.45                                                  Arg-69    NH1      C8   3.28                                                                     C2'  3.48                                                                     O2'  3.33                                                                     O3   2.85                                                                     O6   3.25                                                  Arg-69    NH2      N7   3.16                                                                     C8   3.24                                                                     O3   3.18                                        S5        Gly-58    CA       O2   2.57                                                  Gly-58    C        O2   3.40                                                  Ser-62    OG       N7   2.80                                        ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Characterization of PT analogs                                                            Concentration of PT analog                                                    by ELISA μg/mL   Mean                                                                   Polyclonal Residual                                                           antibody   toxicity                                  PT Analog     Fetuin Capture                                                                           capture    (%)                                       ______________________________________                                        S2 Lys-83 → Ala                                                                      0.4        1.03       86                                                      0.3        0.87                                                               0.38       0.85                                                               0.33       1.74                                                 S2 Arg-125 → Ala                                                                     0.1        1.16       28                                                      0.15       0.91                                                               0.87       8.05                                                               0.33       3.37                                                               0.36       3.37                                                 S3 Arg-125 → Ala                                                                     0.25       4.95       47                                                      1.20       9.50                                                               1.00       6.90                                                               0.82       1.74                                                               0.82       5.77                                                 Wild-type     0.4        0.6        100                                                     0.75       2.54                                                               1.10       4.48                                                 ______________________________________                                    

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 46                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Val Tyr Arg Tyr Asp Ser                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Phe Val Ser Thr Ser Ser Ser Arg Arg Tyr Th - #r Glu                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Phe Ile Gly Tyr Ile Tyr Glu Val Arg                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ser Glu Tyr Leu Ala His Arg                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Asn Ile Arg Arg Val                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Leu Tyr Arg Ala Asp Ser                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Tyr Val Ser Thr Ser Leu Ser Leu Arg Ser Al - #a His                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Ser Thr Tyr Tyr Ile Tyr Val Ile Ala                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Gln Glu Val Ser Ala Leu Gly                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Gln Ile Tyr Gly Trp                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Gly Tyr His Gly Thr Phe                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Al - #a Tyr                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Arg Asn Gly Ala Leu Leu Arg Val Tyr                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Leu Glu Thr Ile Leu Gly Trp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Val Val Ile Pro Ser                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Ser Tyr His Gly Thr Lys                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Al - #a Ala                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Lys Ala Gly Gly Val Val Lys Val Thr                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Val Glu Tyr Ile Asn Asn Trp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Ser Val Glu Leu Glu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 69 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Thr Pro Asp Cys Val Thr Gly Lys Val Glu Ty - #r Thr Lys Tyr Asn Asp         #                15                                                           - Asp Asp Thr Phe Thr Val Lys Val Gly Asp Ly - #s Glu Leu Phe Thr Asn         #            30                                                               - Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Al - #a Gln Ile Thr Gly Met         #        45                                                                   - Thr Val Thr Ile Lys Thr Asn Ala Cys His As - #n Gly Gly Gly Phe Ser         #    60                                                                       - Glu Val Ile Phe Arg                                                         65                                                                            - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 104 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Gln Pro Ala Thr Asp His Tyr Tyr Ser Asn Va - #l Thr Ala Thr Arg Leu         #                15                                                           - Leu Ser Ser Thr Asn Ser Arg Leu Cys Ala Va - #l Phe Val Arg Ser Gly         #            30                                                               - Gln Pro Val Ile Gly Ala Cys Thr Ser Pro Ty - #r Asp Gly Lys Tyr Trp         #        45                                                                   - Ser Met Tyr Ser Arg Leu Arg Lys Met Leu Ty - #r Leu Ile Tyr Val Ala         #    60                                                                       - Gly Ile Ser Val Arg Val His Val Ser Lys Gl - #u Glu Gln Tyr Tyr Asp         #80                                                                           - Tyr Glu Asp Ala Thr Phe Glu Thr Tyr Ala Le - #u Thr Gly Ile Ser Ile         #                95                                                           - Cys Asn Pro Gly Ser Ser Leu Cys                                                         100                                                               - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 104 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Gln Pro Ala Ala Asp His Tyr Tyr Ser Lys Va - #l Thr Ala Thr Arg Leu         #                15                                                           - Leu Ala Ser Thr Asn Ser Arg Leu Cys Ala Va - #l Phe Val Arg Asp Gly         #            30                                                               - Gln Ser Val Ile Gly Ala Cys Ala Ser Pro Ty - #r Glu Gly Arg Tyr Arg         #        45                                                                   - Asp Met Tyr Asp Ala Leu Arg Arg Leu Leu Ty - #r Met Ile Tyr Met Ser         #    60                                                                       - Gly Leu Ala Val Arg Val His Val Ser Lys Gl - #u Glu Gln Tyr Tyr Asp         #80                                                                           - Tyr Glu Asp Ala Thr Phe Gln Thr Tyr Ala Le - #u Thr Gly Ile Ser Leu         #                95                                                           - Cys Asn Pro Ala Ala Ser Ile Cys                                                         100                                                               - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 110 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Asp Val Pro Tyr Val Leu Val Lys Thr Asn Me - #t Val Val Thr Ser Val         #                15                                                           - Ala Met Lys Pro Tyr Glu Val Thr Pro Thr Ar - #g Met Leu Val Cys Gly         #            30                                                               - Ile Ala Ala Lys Leu Gly Ala Ala Ala Ser Se - #r Pro Asp Ala His Val         #        45                                                                   - Pro Phe Cys Phe Gly Lys Asp Leu Lys Arg Pr - #o Gly Ser Ser Pro Met         #    60                                                                       - Glu Val Met Leu Arg Ala Val Phe Met Gln Gl - #n Arg Pro Leu Arg Met         #80                                                                           - Phe Leu Gly Pro Lys Gln Leu Thr Phe Glu Gl - #y Lys Pro Ala Leu Glu         #                95                                                           - Leu Ile Arg Asn Val Glu Cys Ser Gly Lys Gl - #n Asp Cys Pro                 #           110                                                               - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 99 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Gly Leu Pro Thr His Leu Tyr Lys Asn Phe Th - #r Val Gln Glu Leu Ala         #                15                                                           - Leu Lys Leu Lys Gly Lys Asn Gln Glu Phe Cy - #s Leu Thr Ala Phe Met         #            30                                                               - Ser Gly Arg Ser Leu Val Arg Ala Cys Leu Se - #r Asp Ala Gly His Glu         #        45                                                                   - His Asp Thr Trp Phe Asp Thr Met Leu Gly Ph - #e Ala Ile Ser Ala Tyr         #    60                                                                       - Ala Leu Lys Ser Arg Ile Ala Leu Thr Val Gl - #u Asp Ser Pro Tyr Pro         #80                                                                           - Gly Thr Pro Gly Asp Leu Leu Glu Leu Gln Il - #e Cys Pro Leu Asn Gly         #                95                                                           - Tyr Cys Glu                                                                 - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 93 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Glu Tyr Arg Asn Thr Gln Ile Tyr Thr Ile As - #n Asp Lys Ile Leu Ser         #                15                                                           - Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Me - #t Val Ile Ile Thr Phe         #            30                                                               - Lys Ser Gly Glu Thr Phe Gln Val Glu Val Pr - #o Gly Ser Gln His Ile         #        45                                                                   - Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Ly - #s Asp Thr Leu Arg Ile         #    60                                                                       - Thr Tyr Leu Thr Glu Thr Lys Ile Asp Lys Le - #u Cys Val Trp Asn Asn         #80                                                                           - Lys Thr Pro Asn Ser Ile Ala Ala Ile Ser Me - #t Lys Asn                     #                90                                                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Asn Lys Thr Arg Ala Leu                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Ser Gly Asp Leu Gln Glu Tyr Leu Arg His Va - #l Thr Arg                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Ser Ile Phe Ala Leu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Asp Gly Thr Tyr Leu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Tyr Gly Gly Val Ile Lys Asp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Thr Thr Phe Cys Ile Met                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Asn Gly Thr Arg Ala Leu                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Asn Ala Glu Leu Gln Thr Tyr Leu Arg Gln Il - #e Thr Pro                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Ser Ile Tyr Gly Leu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Asp Gly Thr Tyr Leu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - Tyr Gly Gly Ile Ile Lys Asp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - Glu Thr Phe Cys Ile Thr                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                - Leu Arg Gly Thr Val Ala                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - Asn Ala Glu Glu Asn Lys Ala Ile Gln Glu Va - #l Ala Lys                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - Thr Ser Ala Phe Leu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                - Cys Val Thr Ile Val                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                - Asp Asn Gly Leu Trp Asn Asp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                - Thr Ala Val Cys Glu Phe                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - Ala Met Ala Ala Trp Ser Glu Arg Ala Gly                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                - Val Ala Ser Ile Val Gly Thr Leu Val Arg Me - #t Ala Pro Val Ile Gly         #                15                                                           - Ala Cys Met Ala                                                                         20                                                                __________________________________________________________________________

What we claim is:
 1. A method for producing a modified pertussisholotoxin, comprising:(a) identifying at least one amino acid residue ofthe holotoxin for modification by analyzing a three-dimensionalstructure of crystalline pertussis holotoxin determined by X-raycrystallography in relation to known information concerning proteinstructure and function; (b) effecting mutagenesis of a tox operonencoding the holotoxin to remove or replace a nucleotide sequence codingfor said at least one amino acid residue and to produce a mutant toxoperon; and (c) expressing the mutant tox operon in a Bordetellaorganism to produce the modified holotoxin.
 2. A method for producing amodified form of at least a portion of pertussis holotoxin,comprising:(a) forming a crystalline complex between at least a portionof pertussis holocoxin and a molecule capable of complexing with theholotoxin, wherein said molecule is selected from the group consistingof an adenine nucleotide, NAD, a substantially non-hydrolysable analogof NAD, a GTP-binding protein, and α-subunit of a GTP-binding proteinand a C-terminal fragment of a α-subunit of a GTP binding protein; (b)determining a three-dimensional structure of the comnplex by X-raycrystallography; (c) analysing the structure to identify at least oneamino acid residue of the at least a portion of pertussis holatoxininteracting with the molecule; (d) effecting mutagensis of a nucleotidesequence encoding the at least a portion of the pertussis holotoxin toremove or replace a codon for the at least one amino acid and/or toinsert at least one codon adjacent the codon for the at least one aminoacid to produce a mutant nucleotide sequence; and (e) expressing themutant nucleotide sequence to produce the modified form of at least aportion of pertussis holocoxin.
 3. The method of claim 2, wherein the atleast a portion of pertussis holotoxin is pertussis holotoxin or ananalog thereof and the step of expressing the mutant nucleotide sequenceis effected in a Bordetella organism.
 4. The method of claim 2, whereinthe at least a portion of pertussis holotoxin is a subunit, a portion ofa subunit or a combination of subunits.
 5. The method of claim 2,wherein the at least one additional codon is inserted adjacent the3'-codon of the nucleotide sequence encoding the S1 subunit of pertussisholotoxin.
 6. The method of claim 5, wherein said at least oneadditional codon codes for at least one negatively-charged amino acid.7. The method of claim 6, wherein said negatively-charged amino acid isAsp or Glu.
 8. The method of claim 1 or 2, wherein the at least oneidentified amino acid residue contributes to a biological activity ofthe holotoxin selected from the group consisting of toxicity, cellbinding, mitogenicity, enzymatic activity and adjuvanticity.
 9. Themethod of claim 3, wherein said at least one amino acid residue isselected from the group consisting of any one of the following aminoacid residue:

    ______________________________________                                        Subunit             Residues                                                  ______________________________________                                        S1                  Phe-23                                                                        Ser-48                                                                        Val-51                                                                        Gln-127                                                                       Leu-131                                                                       Gly-199                                                                       Ala-200                                                                       Phe-235                                                   S2                  His-15                                                                        Gln-16                                                                        Trp-52                                                                        Glu-66                                                                        Asp-81                                                                        Leu-82                                                                        Lys-83                                                                        Ser-104                                                                       Arg-125                                                                       Ser-147                                                                       Arg-150                                                                       Lys-151                                                   S3                  Gln-15                                                                        Gln-16                                                                        Tyr-82                                                                        Arg-83                                                                        Ser-104                                                                       Arg-125                                                                       Arg-150                                                                       Arg-151                                                   S4                  Asp-1                                                                         Tyr-4                                                                         Gly-60                                                                        Ser-61                                                                        Glu-65                                                                        Arg-69                                                                        Thr-88                                                                        Pro-93                                                                        Asp-54                                                                        Thr-51                                                    ______________________________________                                    


10. The method of claim 8, wherein said at least one amino acid residueis substituted by an amino acid residue as follows:

    ______________________________________                                        Subunit       Residues     Replacement                                        ______________________________________                                        S1            Phe-23       Asp or Glu                                                       Ser-48       Ala                                                              Val-51       Ile                                                              Gln-127      Ala or Asp                                                       Leu-131      Lys or Arg                                                       Gly-199      Val or Gln                                                       Ala-200      Ile                                                              Phe-235      Glu                                                S2            His-15       Ala or Thr                                                       Gln-16       Ala or Thr                                                       Trp-52       Val                                                              Glu-66       Ala or Lys                                                       Asp-81       Ala or Ser                                                       Leu-82       Ala or Glu                                                       Lys-83       Glu                                                              Ser-104      Ala                                                              Arg-125      Ala                                                              Ser-147      Thr                                                              Arg-150      Ser                                                              Lys-151      Ser                                                S3            Gln-15       Ala or Thr                                                       Gln-16       Ala or Thr                                                       Tyr-82       Ala or Val                                                       Arg-83       Glu                                                              Ser-104      Ala                                                              Arg-125      Ala                                                              Arg-150      Ser                                                              Arg-151      Ser                                                S4            Asp-1        Ala                                                              Tyr-4        Ala or Val                                                       Gly-60       Val                                                              Ser-61       Ala                                                              Glu-65       Ala                                                              Arg-69       Ala                                                              Thr-88       Val                                                              Pro-93       Ala                                                              Asp-54       Glu                                                              Thr-51       Tyr                                                              Thr-55       Tyr                                                              Gly-58       Val                                                S5            Ser-62       Ala                                                ______________________________________                                         
 11. The method of claim 1, wherein said at least one amino acid residue     is located from residue 184 to 203 or from residue 211 to 220 of subunit     S1 of pertussis holotoxin.